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Viral diseases are a threat to bacteria and enormous animals alike. Vaccines are available against several viruses. However, for some viruses, like ASFV, we still lack vaccines, while for others, like IAV, they are not as effective as we need them to be. To a large extent, this is because we do not fully understand the mechanisms conferring antiviral immunity. To improve our understanding of antiviral immunity, we used a model species that is in many immunological aspects closer to humans than the widely used laboratory mice, pigs. In this thesis, pigs were investigated as a potential biomedical model species for viral respiratory infections in humans and as a natural host for viral infections. Both approaches provide valuable insights into aspects of porcine immunology that can either be used as the foundation for translational research or for the design of targeted therapeutics and vaccines for pigs.
Insights into fundamental characteristics of the porcine immune system form the basis for translational studies. Paper I pioneered a detailed characterization of porcine iNKT cells. To make pigs and porcine iNKT cells more available for scientific investigations, we established multicolor flow cytometry analysis platforms that allow for a more detailed investigation of these cells than previously possible. We found porcine iNKT cells circulating in peripheral blood to be a rare population among CD3+ lymphocytes that displays a pre-activated effector state and can be divided into at least three functional subsets. Upon antigenic activation, they proliferated rapidly, secreted pro-inflammatory cytokines, and exerted cytotoxicity. Moreover, we provided first evidence for a role of iNKT cells in porcine IAV and ASFV infections, which we investigated in more detail in paper IV. Central characteristics, i.e., phenotype and functional properties, exhibit a high degree of similarity between humans and pigs. Moreover, differences between human and murine iNKT cells are more pronounced than between humans and pigs.
Based on the results obtained in paper II, the established biomedical model could be used for further studies of infectious respiratory diseases. IAV infections pave the way for secondary co-infections with increased morbidity and lethality. These bactoviral co-infections are a threat to both pigs and humans. The shared susceptibility as well as homologies on the physiological and immunological level make pigs exceptionally suitable animal models for studies of these infections. Paper I and II can also be interpreted under translational aspects. Activation of iNKT cells in porcine vaccination studies showed promising results. Based on these and our findings, this might be a suitable approach for humans as well. Along with other studies, our results suggest that pigs might be a well-suited large animal model for research in infectious diseases. This is true especially for respiratory infections, such as seasonal IAV infections, for which pigs are natural hosts and contribute to viral spread and emergence as “mixing vessels”, which can result in pandemic strains like H1N1pdm09. We could show that porcine iNKT cells as well as the antiviral responses of cTC against H1N1pdm09 in pigs are comparable to human cells and processes. The increased implementation of pigs in basic and applied research might enable an improved translation of scientific knowledge to human and veterinary medicine.
In two further studies, papers III and IV, we investigated T-cell responses during a viral infection, ASF, for which pigs are the only natural hosts. Immune responses were similar after highly and moderately virulent ASFV infection in domestic pigs and wild boar, respectively. However, they differed between both species. Antiviral immunity in domestic pigs was predominantly exerted by αβ T cells, CD8α+ and DP αβ T cells, while the response in wild boar was dominated by γδ T cells, mainly CD8α+ effector cells. Since wild boar show a higher disease severity and lethality, even during infection with moderately virulent ASFV “Estonia2014”, a shift to γδ T cells seems to be detrimental. In contrast, domestic pigs survive infections with moderately virulent ASFV “Estonia2014”, which indicates that CD8α+ or DP αβ T cells confer protection at least in infections with non-highly virulent ASFV strains. Interestingly, in paper V we found higher and prolonged inflammation in domestic pigs, correlating with increased T-cell influx. However, histopathological analyses revealed no direct explanation for the differences in disease progression and lethality in domestic pigs and wild boar. These findings require further studies to elucidate the underlying mechanisms.
The lack of basic data about immunological differences between domestic pigs and wild boar hampers attempts to understand immunity against ASFV. We found differences between both suid subspecies already at steady state and even more prominent during ASFV infections in papers III-V. Most apparently, T-cell responses in wild boar were heavily biased towards γδ T cells, while immune responses in domestic pigs were based on αβ T cells. However, information about even basic characteristics, like the composition, phenotypes, and functional qualities of wild boar’s immune system, is missing. Therefore, essential baseline data must be obtained in order to adequately assess changes in future studies.
Analyses like these reveal major advantages of pigs as a biomedical model. On the one hand, similar to conventional model species, researchers can investigate every tissue at any desired time. Tissue from human patients is often scarce or not at all available, so models that can be investigated at specific times after infection are needed. On the other hand, results obtained in pigs are more comparable to humans than data from murine studies. Moreover, pigs are susceptible to similar pathogens as humans and experimental infections can be investigated without the need for major genetic manipulations. However, there are also limitations of the porcine model system. Analysis tools are not as advanced as they are for mice, especially in terms of availability of mAbs or genetically modified organisms. Still, given the major advantages that become more and more obvious, efforts should be made to make pigs more applicable for basic and translational research. In addition, findings derived from pigs can be used for the species itself. Pigs are a major livestock species and new treatments, or vaccines could also be used for them. Therefore, this research could eventually also improve animal welfare.
In summary, the presented thesis significantly enhanced our knowledge of porcine immune processes for cTC in general and iNKT cells in particular. Results were obtained both at steady state and in the context of IAV and ASFV infections, and thus, made pigs more available as a model for future research. The use of multicolor flow cytometry provided a broad overview of the ongoing immune reactions and enables further, more wide-ranging studies that can also address open questions in even more complex infection scenarios.
Die Segmentierung des Influenza-A-Virusgenoms und die damit verbundene Möglichkeit des Reassortments sind von großer Bedeutung für die Adaptation an einen neuen Wirt und die Entstehung von Pandemien. So wurden verschiedene Influenza-Pandemien des letzten Jahrhunderts durch ein humanes Virus ausgelöst, das mindestens das Hämagglutinin (HA) eines aviären Influenza-A-Virus übernommen hatte. Mit Hilfe der Reversen Genetik ist es möglich, den Austausch von Segmenten zwischen verschiedenen Influenza-Viren detailliert zu untersuchen. Es können Erkenntnisse zur Art und Häufigkeit von Reassortments gewonnen werden, die zu einem besseren Verständnis der Entstehung potentiell pandemischer Viren führen. In der vorliegenden Arbeit wurde zunächst die revers-genetische Methode der target-primed plasmid amplification erfolgreich zur Generierung des vollständigen Plasmidsatzes des c-DNA-Genoms des humanen Influenza-A-Virus A/Denver/57 (H1N1) angewendet. Die Funktionsfähigkeit des klonierten Plasmidsatzes konnte durch Kotransfektionsexperimente gezeigt werden. In einem nächsten Schritt wurde eine PCR etabliert, welche die simultane Amplifikation der cDNA der NS-, M-, NA- und NP-Segmente eines beliebigen Influenza-A-Virus ermöglicht und damit die zeitaufwendige Klonierung der Influenza-Gene deutlich beschleunigt. Dafür wurde ein Primerpaar entwickelt, das an die konservierten Termini der Gensegmente bindet, aber am 3-Ende um die Gen-spezifischen Nukleotide verkürzt ist. Mit den entwickelten Primern kann außerdem das komplette Neuraminidase-Gen unabhängig vom Subtyp amplifiziert werden. In einer Reassortmentstudie konnte die etablierte PCR zur effektiven Genotypisierung eingesetzt werden. Gegenstand der Reassortmentstudie war die Frage, ob sich ältere und jüngere aviäre Stämme in ihrer Fähigkeit, ihr HA an einen humanen Stamm abzugeben, unterscheiden. Außerdem sollte untersucht werden, welche Segmente mit dem aviären HA kosegregieren. Dafür wurden Doppelinfektionsversuche mit jeweils einem der beiden aviären Influenza-Viren A/Duck/Ukraine/1/63 (H3N8) (DkUkr63) bzw. A/Mallard/Germany/Wv64-67/05 (H3N2) (MallGer05) und dem humanen Influenza-Virus A/Hongkong/1/68 (H3N2) (Hk68) durchgeführt. Das Einführen einer Elastase-abhängigen HA-Schnittstelle in das humane Virus Hk68 ermöglichte eine effektive Selektion von Reassortanten mit aviärem HA. Unter den jeweils 21 untersuchten Plaqueisolaten gab es 16 (DkUkr63) bzw. 18 (MallGer05) Reassortanten, die mindestens das aviäre HA erworben hatten. Geringe Häufigkeiten des Auftretens bestimmter Reassortanten lieferte Hinweise bezüglich Beschränkungen im Reassortment. Bei der Genotypisierung der Plaqueisolate wurden für DkUkr63 sieben und für MallGer05 vierzehn verschiedene Reassortantenspezies gefunden. Dies deutet darauf hin, dass der Austausch der Segmente von DkUkr63 gegenüber denen von MallGer05 stärker eingeschränkt ist. Bemerkenswert war die häufige Kosegregation des NA mit HA beider Vogelviren. Die Wachstumskinetik auf humanen A549-Zellen zeigte darüber hinaus auch eine gute Replikation für alle HA/NA-Reassortanten. Beide Viren geben also ihr HA wie auch ihr NA leicht an einen humanen Stamm ab. Andererseits war die geringe Häufigkeiten von PB2- im Vergleich zu PB1-Reassortanten auffällig. Dies deutet darauf hin, dass der Austausch des PB2-Segments im Vergleich zu PB1 stärker eingeschränkt ist. Eine der am besten replizierenden Reassortanten wies mit PB1, HA und NA von DkUkr63 die gleiche Genkonstellation auf wie das Pandemievirus der Asiatischen Grippe von 1957. Die Auswertung der Plaque-Morphologie ergab, dass die Plaque-Größe der Reassortanten von der Herkunft des NA abhängig ist. Verglichen mit dem eingeschränkten Wachstum des aviären Elternvirus DkUkr63 zeigten die meisten seiner Reassortanten ein besseres Wachstum auf humanen A549-Zellen. Das jüngere aviäre Elternvirus MallGer05 erreicht fast den Endtiter des humanen Hk68 und auch die meisten Reassortanten zeigten mit Hk68 vergleichbare Endtiter. Es wurden aber für beide Vogelviren auch einige Reassortanten gefunden, deren Replikation deutlich vermindert war, was auf eine geringe Kompatibilität der jeweiligen Segmente hindeutet. Sowohl für den älteren als auch für den jüngeren aviären Elternstamm wurden unter den gewählten Selektionsbedingungen verschiedene HA-Reassortanten mit guter Replikationsfähigkeit gefunden. Dementsprechend könnten auch unter natürlichen Bedingungen sogar niedrig pathogene aviäre Influenza-A-Viren mit geringer Adaptation an einen humanen Wirt bei Koinfektionen mit humanen Viren zur Bildung von neuen, potentiell pandemischen Viren beitragen.